This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Communication through speech is an invaluable part of human health, dramatically facilitating self-expression and enriching personal relationships. Fundamental to this process is the planning and sequential performance of vocal sounds. Motor disorders such as apraxia of speech and stuttering can dramatically disrupt vocal sequencing, severely impairing communication and often evoking harsh ridicule and social isolation. In each disorder, symptoms are thought to arise from anomalies in regions of the central nervous system involved in sequencing the performance of individual sounds, but effective therapies will require detailed knowledge of the deficits that accompany each condition. Presently, the neural circuitry and synaptic mechanisms that shape sequencing of human speech remain largely unknown. Neuroimaging studies suggest important roles for premotor and speech-related cortical areas and the basal ganglia in vocal sequencing. However, neuroimaging techniques do not provide sufficient spatial or temporal resolution to describe the underlying events in neuronal circuits, necessitating use of an animal model of human speech. Songbirds provide an established model of human speech. Both speech and birdsong are learned vocalizations characterized by careful regulation of tonality and vocal sequence, and the songbird brain contains a set of discrete structures specialized for song performance and perception. One structure, nucleus HVC, is essential for song production and important in song perception, indicating functionality analogous to human speech-related cortical areas. HVC directs vocal output through separate projections into premotor (HVCRA) and basal ganglia (HVCX) pathways, and HVC activity directs the timing of individual song features. Furthermore, HVC receives synaptic input from brain regions proposed to regulate vocal sequencing, suggesting that HVC may encode not only timing but also sequencing of vocal production. The song of one species, Bengalese finches (BF), contains individual vocal sounds produced with variable sequence in some portions and fixed sequence elsewhere, providing an animal model of variable and fixed sequencing in human speech. Preliminary studies indicate that activity in at least one input to HVC can modify BF song sequence. Thus, studies of HVC and its afferents in BF will enable synaptic-level analysis of how fixed and variable vocal sequences are encoded in premotor and basal ganglia pathways. The proposed project will investigate the activity of identified neurons in HVC and its synaptic afferents during BF performance of natural and artificially manipulated vocal sequences. AIM 1 will alter activity of afferents to HVC to establish which sites are sufficient to induce changes in vocal sequence. AIM 2 will characterize the singing-related activity of individual neurons in those afferent sites, revealing how naturally fixed and variable vocal sequences are encoded by those structures. AIM 3 will characterize the activity of HVCRA and HVCX neurons as birds sing with or without the afferent manipulations used in Aim 1, revealing how acquired alterations of vocal sequencing are encoded in premotor and basal ganglia networks.